Inhibition of IgA production

ABSTRACT

The production of IgA is selectively inhibited by orally administering 15-deoxyspergualin or pharmacologically acceptable salts thereof, thus preventing and treating IgA-associated immunological diseases such as IgA nephropathy.

TECHNICAL FIELD

The present invention relates to suppression of IgA production, morespecifically, to selective suppression of IgA production for preventingand treating immunological disorders caused by over-production of IgAantibodies in a human and an animal.

BACKGROUND ART

Immunoglobulins, which include an antibody and a protein structurallyand functionally related therewith, are classified into five classes(IgA, IgD, IgE, IgM and IgG) based on their functional properties. Amongthese, IgAs are divided into two subclasses, i.e., serum IgA andsecretory IgA (IgA1 and IgA2). The L-chains of the IgA1 are covalentlybound to H-chains. On the other hand, the L-chains of the IgA2 are boundeach other through S-S bonds instead of being bound to H-chains. 90% ofthe serum IgA is IgA1, whereas 60% of the secretory IgA is IgA2.

Sites of IgA production are present in submucosal plasma cells in, forexample, a tunica propria of a mucous membrane of a digestive tract, ina salivary gland, in a mammary gland and the like. In a tunica propriaof a mucous membrane of a digestive tract in a human, the number ofIgA-producing cells is much greater than that of IgG-producing cells inthe ratio about 20:1, in contrast to the ratio 1:3 (IgA:IgG) in a lymphnode or a spleen. The IgA in mucosal secretions is produced as a dimerhaving one J-chain component and accompanied by a secretory piece (SC),which is only a little in the serum IgA. The secretory piece is added tothe dimeric IgA molecule while it comes out from submucosal plasma cellsin an intestine or a respiratory tract to mucosal secretions.

The production of an antibody in an organism is induced by stimulationwith a certain antigen. For example, oral administration of a strain ofan enterobacterium, Bifidobacterium longum, has been reported toincrease the total amount of IgA in feces.

A substance capable of non-specifically stimulating antibody producingcells to deal with an antigen more effectively is often called anadjuvant. For example, cholera toxin, which is a causal toxin ofdiarrhea produced by Vibrio cholerae, is known to act on a mucousmembrane of a small intestine and alter the ionic permeability of themembrane. The alteration results in an excretion of a large amount ofelectrolytes and water from the small intestine to cause diarrhealconditions. Cholera toxin B subunit, which is a detoxified componentprepared by removing the substantial portion of the toxin, is known tobe able to elicit an immune response which promotes IgA antibodyproduction (to be able to induce IgA) after penetration into a mucosalmembrane of a small intestine, and thus serve as an adjuvant.

IgG, which is about 65% of serum immunoglobulins (Ig) in humans,consists of antibodies against almost all of the antigens and plays animportant role in systemic protective immunity. On the other hand, IgAplays an important role in local immune reaction. The secretory IgA inmucosal secretions inhibits the binding of a highly pathogenicmicroorganism or an allergen to a mucous membrane. Therefore, IgA notonly prevents an infection but also prevents a component in foods thatmay act as an allergen from passing through a digestive tract wall bybinding to it.

For example, in case where an extracellular toxin is secreted frommicrobial cells, the biological defense by antibodies depends on thedirect action of antibodies bound to the surface of the microorganism.Thus, antibodies can exert various effects by direct binding to amicroorganism.

However, the IgA is also known to act pathologically on a living body.For example, IgA nephropathy is an immunological disorder that is causedby excess immune reactions of IgA in response to an antigen and bydeposition of immune complexes mainly containing IgA onto a glomerulusof a kidney. It is believed that the onset of the nephropathy is causedby long-term high IgA antibody titers. The titer of IgA antibody inblood from a patient with IgA nephropathy is quite higher than that in ahealthy and normal individual. However, it has not been demonstratedwhether the IgA involved in the formation of the immune complex is ofthe serum type from sites other than a mucous membrane (spleen, bonemarrow, peripheral blood, etc.) or of the secretory type from a mucousmembrane (digestive tract, respiratory apparatus, etc.).

It is suspected that the causal agent of the immune reaction is anantigenic stimulus mainly in an upper airway and a digestive tract.Candidate antigens include foods (e.g., gluten, milk, soybean), bacteria(e.g., Haemophilus parainfluenzae), viruses (e.g., Cytomegalovirus,Adenovirus, Epstein-Barr (EB) virus) [Tomino, Y., Bio. Clinica,12(6):375-379 (1997)]. For example, it has been demonstrated that acomponent of Haemophilus parainfluenzae (HP) and an IgA-type anti-HPantibody are present in the glomerulus and serum in a patient with IgAnephropathy [Suzuki, S., Nakatomi, Y., Sato, H. et al., Journal ofAllergy Clinical Immunology, 96:1152-1160 (1995)].

Although hypotheses concerning the cause of the IgA nephropathy and themechanism of its development have been proposed as described above, manyof them are still unclear. There is currently no specific therapy forthe IgA nephropathy. Thus, a dietetic therapy or a pharmacotherapy isused. The dietetic therapy uses a low salt diet or a low protein diet.The pharmacotherapy uses an antiplatelet for suppressing bloodcoagulation in the glomerulus, an angiotensin converting enzymeinhibitor or a calcium antagonist for suppressing a rise in bloodpressure, or an adrenocorticoid [Sakai, H., Bio. Clinica, 16:372-374(1997)].

Effects of several immunosuppressive agents are currently examined byparenteral administration. However, since many of them systemicallysuppress immunological mechanisms such as IgG production and cellularimmunity in addition to the suppression of the over-production of IgA,there is the high risk of causing a severe side effect. Therefore, suchimmunosuppressive agents have not been widely used clinically yet.

Examples of the immunosuppressive agents include 15-deoxyspergualin(designated as DSG hereinbelow) of formula 1:

Gu—(CH₂)₆—CONHCH(OH)CONH(CH₂)₄NH(CH₂)₃—NH₂

wherein Gu represents a guanidino group. DSG is clinically used as animmunosuppressive agent in an injectable form for renal transplantation.

Alternatively, an effect of suppressing antibody production byparenterally administered DSG has been reported in JP-A 8-40887, JP-A5-238932, Okubo, M. et al., Nephron, 60:336-341 (1992), Makino, M. etal., Immunopharmacology, 14:107-114 (1987), Inoue, K. et al.,Proceedings Japanese Society Nephrology 30th Annual Meeting, pp. 191(1987). The suppression of the production of antibodies of IgE, IgG, IgMand the like has been confirmed therein. However, DSG has not beenreported to selectively suppress IgA.

On the other hand, the use of DSG in an oral composition has beendescribed in Japanese Patent 2610621 and JP-A 8-40887. However,selective suppression of a specified class of antibody has not beendisclosed.

OBJECTS OF INVENTION

One object of the present invention is to provide a useful and novelmeans for preventing and treating immunological disorders caused byover-production of IgA antibodies in a human and an animal.

The other objects a nd advantages of the present invention will beapparent from the description below.

SUMMARY OF INVENTION

The present inventors have confirmed that orally administered DSGeffectively suppresses the production of secretory IgA and serum IgA byusing an animal experimental model which has been made to produce IgA inmucosal tissues due to stimulation by an antigen. Surprisingly, it hasfurther proved that strong systemic immunosuppressive effects which areobserved upon parenteral administration (e.g., intravenousadministration) of DSG such as significant effects of suppressing IgGproduction at the same level as that of effects of suppressing IgAproduction, is not observed. In addition, the present inventors havedemonstrated that the oral administration of DSG to an animal in whichthe IgA production is increased predominantly suppresses IgA amongantibodies to be suppressed by DSG.

Thus, in the present invention, DSG or a pharmacologically acceptablesalt thereof, which is used for a patient in need of selectivesuppression of IgA antibody production, is given by oral administration,which is safer for a living body than conventional methods.

One embodiment of the present invention is a composition for oraladministration to a human or an animal for selectively suppressing IgAproduction containing DSG or a pharmacologically acceptable salt thereofas an active ingredient.

Another embodiment of the present invention is use of DSG or apharmacologically acceptable salt thereof for the manufacture of acomposition for oral administration to a human or an animal forselectively suppressing IgA production.

Yet another embodiment of the present invention is a method forselectively suppressing IgA production in a human or an animal,characterized in that the method comprises orally administering DSG or apharmacologically acceptable salt thereof to a human or an animal inneed of selective suppression of IgA production.

According to the present invention, immune responses, particularly IgAantibody production, in response to an orally ingested antigen aresuppressed, resulting in little or weak systemic immunosuppression andselective suppression of IgA production. Therefore, the presentinvention is useful for preventing and treating IgA-associatedimmunological disorder such as IgA nephropathy.

As used herein, “selective suppression” of IgA production means that IgAantibody production is significantly suppressed, and IgG antibodyproduction and/or delayed hypersensitivity are not significantlysuppressed.

DETAILED DESCRIPTION OF THE INVENTION

The active ingredient contained in the composition for oraladministration for selectively suppressing IgA production according tothe present invention may be either DSG or a pharmacologicallyacceptable salt of the DSG. DSG forms a salt with an acid. The acid forthe formation of the salt may be an inorganic acid or an organic acid aslong as it is pharmacologically acceptable. Preferable inorganic acidsinclude, for example, hydrochloric acid, sulfuric acid, nitric acid andphosphoric acid. Preferable organic acids include, for example, aceticacid, propionic acid, succinic acid, fumaric acid, maleic acid, malicacid, tartaric acid, glutaric acid, citric acid, benzenesulfonic acid,toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid,propanesulfonic acid, asparagic acid and glutamic acid.

Furthermore, in the present invention, one of spergualin-relatedcompounds of formula 2:

Gu—X₀—X₁—A—X₂—CO—X₃

wherein Gu represents a guanidino group, X₀ represents (CH₂)¹⁻⁶, or aphenylene group or CH₂C₆H₄ which may have a substituent, X₁ represents(CH₂)²⁻⁷ or CH═CH, A represents CONH or NHCO; when A is CONH, X₂represents a residue in which an α-amino group and an a-carboxyl groupare removed from an α-amino acid or a residue in which an ω-amino groupand an α-carboxyl group are removed from an ω-amino acid and afunctional group may be presented in the residue; the stereochemistry ofa residue derived from an α- or ω-amino acid having an optically activecarbon is not specifically limited to L-, D- or DL-form; typical andspecific examples include a residue in which an α-amino group and anα-carboxyl group are removed from an α-amino acid such as glycine,α-hydroxyglycine, α-methoxyglycine and serine as well as a residue inwhich an ω-amino group and an α-carboxyl group are removed from an aminoacid such as β-alanine, γ-aminobutyric acid, δ-aminovaleric acid andε-aminocaproic acid; when A is NHCO, X₂ represents a single bond, CH₂NH,CH₂O, or a substituted or unsubstituted lower alkylene group; the loweralkylene group includes, for example, a methylene group, an ethylenegroup and a propylene group, and the substituent thereof includeshalogen such as fluorine, chlorine and bromine, a lower alkoxy groupsuch as a methoxy group and an ethoxy group, and a hydroxyl group; asused herein, the term “lower” used for a substituent means that thesubstituent has 1-6, preferably 1-3 carbons; X₃ representsNH—(CH₂)₄—N(R₀₁)—(CH₂)₃—NH—R₀₂, wherein R₀₁ represents hydrogen or aresidue in which a hydroxyl group is removed from a carboxyl group inα-phenylglycine, and R₀₂ represents hydrogen or a residue in which ahydroxyl group is removed from a carboxyl group in an amino acid or apeptide;

which exhibits a similar effect of suppressing IgA production with thatof DSG or a pharmacologically acceptable salt thereof as described abovemay be used. As used herein, “15-deoxyspergualins (DSG)” andpharmacologically acceptable salts thereof include a compound selectedfrom these spergualin-related compounds represented by formula 2 and apharmacologically acceptable salt thereof.

The spergualin-related compound is a derivative of spergualin isolatedfrom a producer strain of genus Bacillus, which is known to have ananti-tumor activity, an immunopotentiating activity or animmunosuppressive activity depending on the type of the derivative (JP-A58-62152, JP-A 61-129119, JP-A 64-90164). In addition, a method forproducing DSG of formula 1 is disclosed in publications such as JP-B61-23183.

DSG or a pharmacologically acceptable salt thereof which is contained asan active ingredient in the composition for oral administrationaccording to the present invention may be produced according to theknown method as described above or a modification thereof.

The composition for oral administration according to the presentinvention is formulated according to a known method by using DSG or apharmacologically acceptable salt thereof alone or mixing it with anexcipient or a carrier. DSG may be an active stereoisomer or a racemiccompound.

Any pharmacologically acceptable substance may be used as an excipientor a carrier. For example, water, an alcohol, an animal or vegetable oilsuch as soybean oil, peanut oil, sesame oil and mineral oil, orsynthetic oil is used as a liquid carrier. A saccharide such as lactose,maltose and sucrose, an amino acid, a cellulose derivative such ashydroxycellulose, an organic salt such as magnesium stearate, dextran,dextran sulfate, chondroitin sulfate, heparin, gelatin and the like areused as a solid carrier. These solid carries or liquid carriers can beused to prepare a formulation for oral administration such as a tablet,a capsule, a powder, a granule, a solution, a dry syrup or a microsphereformulation. A formulation in a single dosage form is preferable.

Additionally, an acid or an alkali or a suitable amount of buffer may beadded to the composition according to the present invention in order toadjust pH.

Furthermore, it is desirable to add a surfactant such as sodium laurateor glycocholic acid, or βcyclodextrin to the composition for oraladministration according to the present invention in order to increasethe oral absorbability of DSG or a pharmacologically acceptable saltthereof contained as an active ingredient. It is also desirable toprepare a microsphere formulation using biodegradable lactate polymer,lactate-glycolate copolymer or the like in order to promote the uptakefrom a mucous membrane of digestive tract and increase theabsorbability.

Although the content of DSG or a pharmacologically acceptable saltthereof in the composition for oral administration according to thepresent invention varies depending on the formulation, the content isusually 0.1-100% by weight, preferably 1-98% by weight. Generally, atablet, a capsule, a powder or a granule contains 5-100% by weight,preferably 25-98% by weight of the active ingredient.

In the method for selectively suppressing IgA production according tothe present invention, an effective amount of DSG or a pharmacologicallyacceptable salt thereof to suppress IgA production is orallyadministered to a human or an animal in need of selective suppression ofIgA production.

The dose is determined depending on the age, body weight, diseaseconditions, purpose of therapy or the like of a human or an animal as asubject such as a mammal including a pet such as a dog or a cat and adomestic animal. The therapeutic dose for oral administration ispreferably 0.01-5 mg/kg/day or 3-300 mg per adult human (weighing 65 kg)per day. More preferably, the dose is determined such that IgGproduction is not significantly suppressed at the least. Thus, thecomposition for oral administration according to the present inventionis characterized in that it significantly suppresses IgA production evenwith a dose that does not significantly suppress IgG production at theleast.

Certain laboratory animals such as mice grown in a specializedenvironment have not been sensitized immunologically to produce IgA. Theeffect of selectively suppressing IgA production of the composition fororal administration according to the present invention can be confirmedby using an experimental model that is made to produce mucosal IgA andserum IgA by oral administration of an antigen for mucosal immunologicalsensitization. On the other hand, a human or a domestic animal hasalready been subjected to mucosal immunological sensitization due toorally ingested heterologous antigens, for example, in foods, andproduces IgA. Therefore, the composition for oral administrationaccording to the present invention exhibits an effect of selectivelysuppressing the production of mucosal IgA and serum IgA against anorally inoculated antigen in a human or a domestic animal as it does inthe experimental model.

Diseases due to mucosal IgA and/or serum IgA are exemplified by IgAnephropathy, for example. The oral composition according to the presentinvention is useful for treating IgA nephropathy.

The following Examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof. DSG used inExamples is a compound of formula 1 as described above.

EXAMPLE 1

Effect of suppressing secretory IgA production in response to orallyadministered antigen by DSG

An effect of suppressing secretory IgA production by orally administeredDSG was confirmed by measuring titers of anti-cholera toxin IgAantibodies in feces from mice orally given cholera toxin.

First, a cholera toxin solution and a DSG solution were prepared.Cholera toxin (Sigma) was dissolved in 0.2 M NaHCO₃ at a concentrationof 50 μg/ml to obtain the cholera toxin solution. DSG (Takara Shuzo,lyophilized original drug) was dissolved in saline at a concentration of5 mg/ml or 1.25 mg/ml to obtain the DSG solution.

Next, 0.2 ml of the cholera toxin solution was orally administered twice(on day 0 and day 7) to three groups of C57BL/6 mice (seven weeks old,female, five mice per group). Hereinbelow in this Example, the number ofdays is the one counted from day 0 on which the cholera toxin wasadministered for the first time. 0.2 ml/dose of the DSG solution at 5mg/ml, the DSG solution at 1.25 mg/ml or saline was orally administeredfive times (once per day from day 7 to day 11) to the three groups ofmice. The doses of DSG orally administered to the respective groups were50 mg/kg, 12.5 mg/kg and 0 mg/kg, respectively. Feces were collectedfrom the mice of the respective groups on day 7 and day 14. Antibodiesin 0.1 g of the feces were extracted with 1 ml of phosphate bufferedsaline (PBS). The extract is designated as a feces antibody extracthereinbelow. Feces on day 7 were collected before the administration ofDSG.

The titer of IgA antibodies in the feces antibody extracts was measuredby an ELISA method. 50 μl each of a cholera toxin solution at 10 μg/mlin 0.2 M NaHCO₃ was added to Immuno Modules (Nunc), which were allowedto stand at 4° C. for 16 hours to coat the cholera toxin to obtain asolid phase for the ELISA method. After the solid phase was then blockedwith a BSA solution containing 1% (w/v) bovine serum albumin (Sigma)dissolved in PBS, 50 μl each of the feces antibody extracts was addedthereto, then reacted at 37° C. for 1 hour. 50 μl of horseradishperoxidase (HRP)-labeled rabbit anti-murine IgA antibody (Zymed) wasadded and reacted at 37° C. for 1 hour. 50 μl of an ABTS solutioncontaining 2.75 mg/ml of azino bis-ethylbenzothizoline sulfonic acid(ABTS, a substrate for HRP, nacalai tesque) dissolved incitrate-phosphate buffer was then added thereto, reacted at roomtemperature for 15 minutes, and the absorbance at wavelength of 405 nm(OD₄₀₅) was then measured.

The results are shown in Table 1. In Table 1, the increased amount ofsecretory IgA antibody production means the difference between theamount of the IgA production on day 7 and that on day 14. The differenceis expressed by the difference between the OD₄₀₅ value with the fecesantibody extract of day 7 and the OD₄₀₅ value with the feces antibodyextract of day 14 (mean±standard deviation).

TABLE 1 Increased amount of Dose of DSG secretory IgA antibodySignificant (mg/kg) production difference p 0 0.409 ± 0.091 12.5 0.249 ±0.092 <0.05 50 0.082 ± 0.017 <0.0001

As seen from the results in Table 1, the oral administration of DSG at50 mg/kg and 12.5 mg/kg resulted in significant decrease in theincreased amount of secretory IgA production in response to the orallyadministered antigen as compared with that observed for the groupreceived no DSG. These results confirm the effect of suppressing IgAproduction by the orally administered DSG.

EXAMPLE 2

Effect of selectively suppressing IgA production in response to orallyadministered antigen by DSG

The effect of suppressing antibody production by orally administered DSGwas confirmed by measuring the titer of anti-cholera toxin IgA antibodyin feces, as well as the titer of anti-cholera toxin IgA antibody andanti-cholera toxin IgG antibody in blood from a mouse orally receivedcholera toxin.

Cholera toxin was orally administered to five groups of C57BL/6 mice(seven weeks old, female, five mice per group) as described inExample 1. 0.2 ml/dose of one of three DSG solutions or saline wasorally administered ten times (once a day, from day 7 to day 17excluding day 13) to four groups of mice for cholera toxinadministration. The solutions contained DSG at 5 mg/ml, 1.25 mg/ml or0.313 mg/ml dissolved in saline. The doses of DSG orally administered tothe respective groups were 50 mg/kg, 12.5 mg/kg, 3.13 mg/kg and 0 mg/kg,respectively. 0.2 ml/dose of a DSG solution at 0.313 mg/ml wasintraperitoneally administered ten times (once a day, from day 7 to day17 excluding day 13) to the remaining one group of mice for choleratoxin administration. The dose of intraperitoneally administered DSG was3.13 mg/kg.

Feces were collected from mice from the respective groups on day 7 andday 18. Antibodies were extracted with 1 ml of PBS from 0.1 g of thefeces. The extract is designated as a feces antibody extracthereinbelow. The feces of day 7 were collected before the administrationof DSG.

On the other hand, blood was partially collected on day 7 from orbitalvenous plexus of a mouse from each group under etherization and thewhole body blood was collected on day 18. Sera were separated from thethus-obtained blood and diluted 50-folds. The dilution is designated asa blood antibody sample hereinbelow. The blood on day 7 was collectedbefore the administration of DSG.

The titer of IgA antibodies against cholera toxin in the feces antibodyextract or the blood antibody sample was measured by an ELISA method.The measurement was carried out as described in Example 1. The bloodantibody sample was also used to determine the amount of IgG antibodiesagainst cholera toxin produced in blood. The solid phase used for thetitration of antibodies by the ELISA method is the same as that used forthe titration of IgA antibodies by the ELISA method in Example 1.

After the solid phase was blocked with a BSA solution as described inExample 1, 50 μl each of the antibody samples was added thereto, andthen reacted at 37° C. for 1 hour. 50 μl of HRP-labeled rabbitanti-murine IgG antibody (Zymed) was added and reacted at 37° C. for 1hour. 50 μl of a 2.75/ml ABTS solution was then added thereto, reactedat room temperature for 15 minutes, and the absorbance at wavelength of405 nm (OD₄₀₅) was then measured.

The results are shown in Table 2. Table 2 shows a variety of thedifferences between the amount of antibodies produced on day 7 and thaton day 18. The difference is expressed by the difference between theOD₄₀₅ value with the feces antibody extract or the blood antibody sampleof day 7 and the OD₄₀₅ value with the feces antibody extract or theblood antibody sample of day 18 (mean±standard deviation)

TABLE 2 DSG Dose Antibody titer (mg/kg) Secretory IgA Blood IgA BloodIgG 0 0.308 ± 0.075   0.051 ± 0.013 0.493 ± 0.148  3.13 (p.o.) 0.327 ±0.065   0.035 ± 0.012 0.472 ± 0.112  12.5 (p.o.) 0.112 ± 0.116*   0.028± 0.012* 0.570 ± 0.107  50 (p.o.) 0.009 ± 0.054**   0.016 ± 0.020* 0.330± 0.169  3.13 (i.p.) 0.008 ± 0.016***  −0.009 ± 0.009*** 0.034 ± 0.035**

In Table 2, the superscript symbols on the right of numbers, *, ** and***, represent significant differences of P<0.05, P<0.001 and P<0.0001,respectively. Additionally, p.o. represents a group orally administeredwith DSG and i.p. represents a group intraperitoneally administered withDSG.

The effect of suppressing antibody production by intraperitoneallyadministered DSG in feces and blood is very strong. Production of eitherof the antibodies, secretory IgA, blood IgA or blood IgG, was completelysuppressed, suggesting the generation of systemic immunosuppression. Theoral administration suppressed the antibody production of secretory IgAand blood IgA in a dose-dependent manner. The low degree of suppressionof antibody production by the oral administration as compared with thatby the intraperitoneal administration as well as the absence of thesuppression of the IgG production in blood suggest that the systemicimmunosuppression by the administration was slight.

EXAMPLE 3

Suppression of antibody production and delayed hypersensitivity inresponse to subcutaneously administered antigen by DSG.

In contrast to Example 1 and 2, the effect of orally administered DSG onantibody production in response to a subcutaneously administered antigenwas confirmed. Briefly, after ovalbumin was subcutaneously administeredto a mouse, DSG was administered orally. The titer of anti-ovalbumin IgAantibody in murine feces, as well as the titer of anti-ovalbumin IgAantibody and the titer of anti-ovalbumin IgG antibody in blood weremeasured to confirm the effect. Additionally, the effect of DSG ondelayed hypersensitivity against ovalbumin was confirmed. (1)Suppression of antibody production: An ovalbumin suspension forsubcutaneous administration was prepared by mixing ovalbumin (Sigma)with Freund complete adjuvant at a concentration of 200 μg/ml. A DSGsolution was prepared by dissolving DSG in saline at a concentration of5 mg/ml, 1.25 mg/ml or 0.313 mg/ml as described in Example 1.

0.1 ml of the ovalbumin suspension was then subcutaneously administeredtwice (day 0 and day 14) to three groups of C57BL/6 mice (seven weeksold, female, five mice per group). Hereinbelow in this Example, thenumber of days is the one counted from day 0 on which the ovalbumin wasadministered for the first time. 0.2 ml/dose of the DSG solution orsaline was then orally administered ten times (once a day, from day 14to day 24 excluding day 20) to the three groups of mice for ovalbuminadministration. The doses of DSG orally administered to the respectivegroups were 50 mg/kg, 12.5 mg/kg and 0 mg/kg, respectively. 0.2 ml/doseof a DSG solution at 0.313 mg/ml was intraperitoneally administered tentimes (once a day, from day 14 to day 25 excluding day 20) to theremaining one group of mice for ovalbumin administration. The dose ofintraperitoneally administered DSG was 3.13 mg/kg.

Feces were collected from mice from the respective groups on day 14 andday 25. Antibodies in 0.1 g of the feces were extracted with 1 ml ofPBS. The extract is designated as a feces antibody extract hereinbelow.The feces of day 14 were collected before the administration of DSG.

On the other hand, blood was partially collected on day 14 from orbitalvenous plexus of a mouse from each group under etherization and thewhole body blood was collected on day 25. Sera were separated from thethus-obtained blood and diluted 50- or 250-folds. The dilution isdesignated as a blood antibody sample hereinbelow. The blood of day 14was collected before the administration of DSG.

The titer of antibodies against ovalbumin in the feces antibody extractor the blood antibody sample was measured by an ELISA method. Themeasurement was carried out as described in Example 2 for the ELISAmethod except that the antigen used for the solid phase was changed fromcholera toxin to ovalbumin.

The subcutaneous administration of ovalbumin did not increase the amountof IgA antibody in the feces or the blood. On the other hand, thesubcutaneous administration of ovalbumin increased the amount of IgGantibody in the blood. The results for the change in the amount of IgGantibody produced in blood are shown in Table 3. Table 3 shows thedifference between the amount of a variety of antibodies produced on day14 and that on day 25. The difference is expressed by the differencebetween the OD₄₀₅ value with the blood antibody sample of day 14 diluted250-folds and the OD₄₀₅ value with the blood antibody sample of day 25diluted 250-folds (mean±standard deviation).

TABLE 3 Dose of DSG Significant (mg/kg) Blood IgG difference P 0 0.437 ±0.173 12.5 (p.o.) 0.286 ± 0.074 50 (p.o.) 0.125 ± 0.141 <0.05 3.13(i.p.) 0.064 ± 0.080 <0.001

In Table 3, p.o. represents a group orally administered with DSG andi.p. represents a group intraperitoneally administered with DSG.

The administration of DSG at 50 mg/kg significantly suppressed the IgGantibody production in blood, whereas the administration of DSG at 12.5mg/kg did not significantly suppressed it. On the other hand, theintraperitoneal administration of DSG at 3.13 mg/kg suppressed theantibody production in blood more strongly than the oral administrationof DSG at 50 mg/kg did.

The above-mentioned results show that the oral administration of DSGselectively suppresses the antibody production in response to an orallyingested antigen rather than that in response to a parenterallyinoculated antigen.

(2) Suppression of delayed hypersensitivity: 25 μl of an aqueoussolution of ovalbumin (400 μg/ml) was subcutaneously administered to afoot pad of a mouse in each of the experimental groups in Example 3(1)24 days after the ovalbumin administration. Swelling in the foot pad wasmeasured 24 hours after the subcutaneous administration of ovalbumin.The results are shown in Table

TABLE 4 Dose of DSG Swelling in foot pad (mg/kg) (× 10⁻² mm) 0 88.6 ±57.2 12.5 (p.o.) 83.2 ± 41.4 50 (p.o.) 62.0 ± 24.8 3.13 (i.p.) 22.8 ±35.3

No difference was recognized between the swelling in foot pad observedfor a group orally administered with DSG at 50 mg/kg and that observedfor group without administration. On the other hand, the swelling infoot pad observed for a group intraperitoneally administered with DSG at3.13 mg/kg was reduced as compared with that observed for a groupwithout administration. Thus, it was suggested that oral administrationof DSG does not suppress delayed hypersensitivity and does not cause thesystemic immunosuppression due to DSG.

EXAMPLE 4

Preparation of oral composition for suppressing IgA antibody production

Granule

50 parts by weight of DSG, 600 parts by weight of lactose, 330 parts byweight of crystallized cellulose and 20 parts by weight of hydroxyprqpylcellulose were mixed well, compacted using a roll-type compactor (RollerCompactor™), ground and put through a sieve between 16-mesh and 60-meshto obtain granules.

Tablet

30 parts by weight of DSG, 120 parts by weight of crystallized lactose,147 parts by weight of crystallized cellulose and 3 parts by weight ofmagnesium stearate were mixed using a V-type mixer and compressed toobtain tablets (300 mg/tablet).

As described above, the oral composition containing DSG or apharmacologically acceptable salt thereof according to the presentinvention suppresses immune reaction in response to an orally ingestedantigen, particularly IgA antibody production, whereas little or weaksystemic immunosuppression is observed. Therefore, the oral compositioncontaining DSG or a pharmacologically acceptable salt thereof accordingto the present invention can be used to treat and prevent IgA-associatedimmunological diseases such as IgA nephropathy.

What is claimed is:
 1. A method for selectively suppressing IgAproduction in a human or an animal, characterized in that the methodcomprises orally administering 15-deoxyspergualin or a pharmacologicallyacceptable salt thereof to a human or an animal in need of selectivesuppression of IgA production.
 2. The method according to claim 1,wherein said human or animal is one suffering from IgA nephropathy. 3.The method according to claim 1 wherein said 15-deoxyspergualin orpharmaceutically acceptable salt thereof is administered in an amountsufficient to suppress IgA production without significant suppression ofIgC production and/or delayed hypersensitivity.